Haplotype-resolved de novo assembly of the Vero cell line genome

Sène, Marie‐Angélique; Kießlich, Sascha; Djambazian, Haïg; Ragoussis, Jiannis; Xia, Yu; Kamen, Amine

doi:10.1038/s41541-021-00358-9

Cited by 17 publications

(21 citation statements)

References 39 publications

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“…The RNase P gene of Vero CCL81 cells differs from that of human RNase P such that there are four primer-template mismatches; two in each primer (20)(21)(22)(23)(24). This was predicted to greatly decrease the sensitivity of this primer/probe set (25).…”

Section: Results: Droplet Digital Pcr Methodology Validationmentioning

confidence: 99%

The role of sub-genomic RNA in discordant results from RT-PCR tests for COVID-19

Toppings

Oberding

Lin

et al. 2021

Preprint

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Reverse transcription-PCR (RT-PCR) is the standard method of diagnosing COVID-19. An inconclusive test result occurs when one RT-PCR target is positive for SARS-CoV-2 and one RT-PCR target is negative within the same sample. An inconclusive result generally requires retesting. One reason why a sample may yield an inconclusive result is that one target is at a higher concentration than another target. It was hypothesized that concentration differences across targets may be due to the transcription of sub-genomic RNA, as this would result in an increase in the concentration of gene targets near the 3’ end of the SARS-CoV-2 genome. A panel of six digital droplet (dd)PCR assays was designed to quantitate the ORF1, E-gene, and N-gene of SARS-CoV-2. This panel was used to quantify viral cultures of SARS-CoV-2 that were harvested during the eclipse phase and at peak infectivity in such a way as to maximize gene-to-gene copy ratios. Eleven clinical nasopharyngeal swabs were also tested with this panel. In culture, infected cells showed higher N-gene/ORF1 copy ratios than culture supernatants. Both the highest specific infectivity (copies/pfu) and the highest differences between gene targets were observed at 6 hours post-infection (eclipse phase) in infected cells. The same trends in the relative abundance of copies across different targets observed in infected cells was observed in clinical samples, though trends were more pronounced in infected cells. This study showed that a greater copy number of N-gene relative to E-gene and ORF1 transcripts could potentially explain inconclusive results for some RT-PCR tests on low viral load samples. The use of N-gene RT-PCR target(s) as opposed to ORF1 targets for routine testing is supported by this data.Author SummaryThis paper provides insight into a drawback of the standard method of testing for COVID-19 (RT-PCR). The results presented here propose an explanation for why inconclusive results sometimes occur with this method. These results can aid microbiologists in the interpretation of inconclusive test results. These results can also aid in decisions about which COVID-19 test a laboratory should use, as there are a plethora of options available. This is important because this standard testing method will remain a critical tool – globally – for managing the COVID-19 pandemic and any future viral pandemics and epidemics. Thus, it is important to investigate every facet of the testing method. The findings presented here are applicable to any virus which makes sub-genomic transcripts as part of its life cycle. Trends observed in viral cultures are presented alongside the same trends observed in clinical samples. Unlike similar papers in the field, this paper did not strive to develop a new methodology or tool.

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Section: Results: Droplet Digital Pcr Methodology Validationmentioning

confidence: 99%

The role of sub-genomic RNA in discordant results from RT-PCR tests for COVID-19

Toppings

Oberding

Lin

et al. 2021

Preprint

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“…No other viral sequence was detected in the patient-derived samples; however, a small number of sequencing reads associated with Mason-Pfizer monkey virus (formerly Simian retrovirus) or Baboon endogenous virus were observed in the samples from the SARS-CoV-2 isolates. Interestingly, the complete genomes of these two retroviruses have been shown to be inserted into the Vero cell line genome [61]; therefore, the Vero cells used to isolate SARS-CoV-2 were the most likely source of the limited number of retroviral sequences.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the First SARS-CoV-2 Isolates from Aotearoa New Zealand as Part of a Rapid Response to the COVID-19 Pandemic

Harfoot

Lawley

Hernández

et al. 2022

Viruses

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SARS-CoV-2, the virus responsible for the COVID-19 pandemic, has wreaked havoc across the globe for the last two years. More than 300 million cases and over 5 million deaths later, we continue battling the first real pandemic of the 21st century. SARS-CoV-2 spread quickly, reaching most countries within the first half of 2020, and New Zealand was not an exception. Here, we describe the first isolation and characterization of SARS-CoV-2 variants during the initial virus outbreak in New Zealand. Patient-derived nasopharyngeal samples were used to inoculate Vero cells and, three to four days later, a cytopathic effect was observed in seven viral cultures. Viral growth kinetics was characterized using Vero and VeroE6/TMPRSS2 cells. The identity of the viruses was verified by RT-qPCR, Western blot, indirect immunofluorescence assays, and electron microscopy. Whole-genome sequences were analyzed using two different yet complementary deep sequencing platforms (MiSeq/Illumina and Ion PGM™/Ion Torrent™), classifying the viruses as SARS-CoV-2 B.55, B.31, B.1, or B.1.369 based on the Pango Lineage nomenclature. All seven SARS-CoV-2 isolates were susceptible to remdesivir (EC50 values from 0.83 to 2.42 µM) and b-D-N4-hydroxycytidine (molnupiravir, EC50 values from 0.96 to 1.15 µM) but not to favipiravir (>10 µM). Interestingly, four SARS-CoV-2 isolates, carrying the D614G substitution originally associated with increased transmissibility, were more susceptible (2.4-fold) to a commercial monoclonal antibody targeting the spike glycoprotein than the wild-type viruses. Altogether, this seminal work allowed for early access to SARS-CoV-2 isolates in New Zealand, paving the way for numerous clinical and scientific research projects in the country, including the development and validation of diagnostic assays, antiviral strategies, and a national COVID-19 vaccine development program.

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“…Total RNA sequencing (TrueSeq) was performed using Illumina NovaSeq6000 Sprime v1.5, PE100. Following standard quality control, the reads were rst aligned to the recently published Vero cell genome [13] using STAR [24] and the resulting BAM les were sorted by name using SAMtools [25] before read count. Transcripts were quanti ed using featureCounts [26].…”

Section: Differential Gene Expression Analysismentioning

confidence: 99%

“…Recent advances in functional genomics and gene editing paved the way for a better understanding and high-throughput engineering of vaccine production cell lines, thus providing new possibilities for cell line development and vaccine bioprocessing intensi cation. With the recent publication of an annotated assembly of the Vero cell genome [13], we propose a transcriptomics analysis of the differences between adherent and suspension Vero cells developed by Shen et al [11]. This process will help highlight key differentially expressed genes and their impact on the cell's phenotype and their metabolic pathways for a better understanding of the process of adaptation to suspension, thus providing insight for new strategies to successfully adapt Vero cells to suspension cultures and enable streamlined scale-up and industrialization.…”

Section: Introductionmentioning

confidence: 99%

Comparative Transcriptomics Analyses of a Vero Cell Line in Suspension Versus Adherent Culture Conditions

Sène

Xia

Kamen

2021

Preprint

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The Vero cell line is the most used continuous cell line for viral vaccine manufacturing. Its anchorage-dependent use renders scaling-up challenging and operations very labor intensive which affects cost effectiveness. Thus, efforts to adapt Vero cells to suspension cultures have been invested but hurdles such as the long doubling time and low cell viability remain to be addressed. In this study, building on the recently published Vero cell line annotated genome, a functional genomics analysis of the Vero cells adapted to suspension is performed to better understand the genetic and phenotypic switches at play during the adaptation of Vero cells from anchorage-dependent to suspension cultures. Results show a downregulation of the epithelial to mesenchymal transition (EMT) pathway, highlighting the dissociation between the adaptation to suspension process and EMT. Surprisingly, an upregulation of cell adhesion components is observed, notably the CDH18 gene, the cytoskeleton pathway, and the extracellular pathway. Moreover, a downregulation of the glycolytic pathway are balanced by an upregulation of the asparagine metabolism pathway, promoting cell adaptation to nutrient deprivation. A downregulation of the adherens junctions and the folate pathways alongside with the FYN gene are possible explanations behind the currently observed low cell viability and long doubling time.

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Haplotype-resolved de novo assembly of the Vero cell line genome

Cited by 17 publications

References 39 publications

The role of sub-genomic RNA in discordant results from RT-PCR tests for COVID-19

The role of sub-genomic RNA in discordant results from RT-PCR tests for COVID-19

Characterization of the First SARS-CoV-2 Isolates from Aotearoa New Zealand as Part of a Rapid Response to the COVID-19 Pandemic

Comparative Transcriptomics Analyses of a Vero Cell Line in Suspension Versus Adherent Culture Conditions

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